This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. It is well recognized that the Fourier amplitudes of cryo-EM data are not as accurate as the corresponding phases. In general, low resolution amplitudes tend to be overweighted and high resolution amplitudes tend to be underweighted. The early work of Crowther showed that sharpening a map using a Gaussian function made the helices more pronounced in their 7.4 [unreadable] map of the hepatitis B capsid core (B[unreadable]ttcher et al., 1997). A similar approach has been adopted in many of the reconstruction maps (e.g. (Zhang et al., 2008b)). However, the actual B factor parameter used for sharpening those maps was somewhat ad hoc. In the EMAN procedure, when we merge the data in 2-D to make class-averages, we apply a Wiener filter (Ludtke et al., 2004). When 3-D reconstruction of these class-averages is performed, this Wiener filter is no longer fully accurate due to the additional averaging that occurs during reconstruction. This results in a moderate overfiltration of EMAN reconstructions before post-processing, even if all of the CTF parameters have been determined accurately. Similarly, the MPSA algorithm also includes a weighting factor of the experimental B factor for each particle image when they are merged through a modified EMAN 3D reconstruction program (Liu et al., 2007b). To compensate, we generally perform a refiltering (sharpening) of the map prior to final interpretation and publication. We are assessing different approaches as how to sharpen the map to bring out the hidden features.